Chemical Constituents, Biological Activities, and Proposed Biosynthetic Pathways of Steroidal Saponins from Healthy Nutritious Vegetable—Allium

Allium is a common functional vegetable with edible and medicinal value. Allium plants have a special spicy taste, so they are often used as food and seasoning in people’s diets. As a functional food, Allium also has abundant biological activities, some of which are used as drugs to treat diseases. By consuming Allium on a daily basis, people can receive active compounds of natural origin, thereby improving their health status and reducing the likelihood of disease. Steroidal saponins are important secondary metabolites of Allium, which are formed by the steroidal aglycone group and sugar. Steroidal saponins have various physiological activities, such as hypoglycemic, antiplatelet aggregation, anti-inflammatory, antitumor, antimicrobial, and enzyme activity inhibition, which is one of the key reasons why Allium has such significant health benefits. The structural diversity and rich biological activities of steroidal saponins make Allium important plants for both food and medicine. In this paper, the chemical structures, biological activities, and structure–activity relationships of steroidal saponins isolated from Allium are reviewed, and the biosynthetic pathways of some key compounds are proposed as well, to provide a molecular reference basis based on secondary metabolites for the health value of Allium.


Introduction
Supplementing nutrition from the diet is a guarantee for human bodies to maintain health. Allium plays an indispensable role in people's diets, whether it is directly eaten as a vegetable or pickled condiments. The unique taste of natural Allium enhances people's appetite, and its abundant biological activities bring people health and nutrition. A. sativum (garlic), for example, is a nutritious vegetable that is widely used as a condiment throughout the world. Fresh garlic bulbs contain about 65% water, 28% carbohydrates, 2.3% organic sulfur compounds, 2% protein, and 1.2% free amino acids (e.g., arginine, glycine, and cystine). Garlic contains 146 kcal/100 g of edible parts. It is also a rich source of vitamin C. There are 10-78.8 mg of this vitamin in 100 g of the edible parts of the product. Garlic also contains minerals-relatively high amounts of potassium, iron, and phosphorus (373-1367 mg, 1.5-13 mg, and 88-522 mg, respectively, in 100 g of the product) [1]. Allium plants usually have medicinal value, which can prevent and treat diseases. A. sativum is believed to have antibacterial, antioxidant, hypotension, and other effects and is used to treat influenza and hypertension.

Furostane Saponins/Sapogenins
Furostane saponins usually have a saturated sapogenin, and its F ring is split. Some furostane saponins form double bonds at C-5(6), C-20(22), C-22 (23), and C-25 (27), or carbonyl groups at C-2, C-6, and C-12. The C-22 position of furostane saponins has α and β configurations. The sugar chains are often attached to C-3, C-26, C-2, and C-1. C-26 is mostly attached to monosaccharides and a few to disaccharides, in particular, C-26 of compound 73 isolated from the bulbs of A. karataviense is linked to trisaccharide [25]. The chemical structures of furostane saponins isolated from Allium in recent years are shown in Figure 1.

Spirostane Saponins/Sapogenins
Most spirostane saponins have a saturated spirostane skeleton, except that some saponins form carbonyl groups at C-2, C-3, C-6, C-12, or double bonds at C-5(6) and C-25 (27). Notably, the A ring of compound 252 is aromatized, compound 249 forms a cyclohexenone structure at the A ring, compounds 250 and 251 form a cyclohexadienone structure at the A ring [73], and compound 253 forms an oxygen bridge between C-2 and C-5 [74]. The sugar moiety is commonly linked to C-2, C-3, and C-24, while compound 164 forms a glycosidic bond at C-27 [53], and compound 181 forms a glycosidic bond at C-6 [59]. The chemical structures of spirostane saponins isolated from Allium in recent years are shown in Figure 2.

Cholestane Saponins/Sapogenins
Cholestane saponins, also known as open-chain saponins, usually have double bonds at C-5 (6) and are oxidized at C-1, C-3, C-16, and C-22, except that compound 267 has no double bond at C-5(6) [5], and the C-1 of compounds 261, 265, and 266 are not oxidized [25,29]. This class of compounds usually forms glycosidic bonds at C-1, C-3, and C-16. The chemical structures of cholestane saponins isolated from Allium in recent years are shown in Figure 3.

Other Types of Steroidal Saponins/Sapogenins
In addition to the three main types mentioned above, there are some steroidal saponins with rare skeletons. Compounds 271 and 274 are derived from pregnane [11,26]. Compounds 269 and 270 have an open A-ring and form a lactone ring at C-5 and C-6 [69]. Compounds 272 and 273 form a lactone ring structure at the E ring [13]. The chemical structures of other types of saponins isolated from Allium in recent years are shown in

Other Types of Steroidal Saponins/Sapogenins
In addition to the three main types mentioned above, there are some steroidal saponins with rare skeletons. Compounds 271 and 274 are derived from pregnane [11,26]. Compounds 269 and 270 have an open A-ring and form a lactone ring at C-5 and C-6 [69].

Biological Activities of Allium Steroidal Saponins
Now it is generally believed that Allium exerts good biological activities due to the presence of organosulfur compounds and steroidal saponins. However, sulfur-containing compounds are much less stable than steroidal saponins, so it is of certain significance to study the biological activities of steroidal saponins of Allium. Modern pharmacological studies have demonstrated the hypoglycemic, antiplatelet aggregation, anti-inflammatory and other activities of these components through in vitro and in vivo experiments, confirming the medicinal value of Allium.

Hypoglycemic Effect
Visfatin is an insulin-mimetic adipocytokine that acts synergistically with insulin to enhance glucose uptake in vivo and in vitro and to inhibit the breakdown of liver glycogen into glucose, with insulin resistance-reducing and antidiabetic effects. Compound 133 isolated from A. macrostemon Bunge can increase the transcription of visfatin partly through the p38 MAPK pathway in differentiated 3T3-L1 adipocytes, which in turn increases the mRNA expression of visfatin and enhances the synthesis and secretion of the visfatin protein, thus having a hypoglycemic effect [47].

Antiplatelet Aggregation Effect
Adenosine diphosphate (ADP) can cause platelet aggregation through ADP receptors on platelet membranes, leading to thrombosis. Thrombus often causes angina pectoris, myocardial infarction, cerebral infarction, pulmonary embolism, and other acute diseases. A. macrostemon Bunge is a common traditional Chinese medicine used to treat

Biological Activities of Allium Steroidal Saponins
Now it is generally believed that Allium exerts good biological activities due to the presence of organosulfur compounds and steroidal saponins. However, sulfur-containing compounds are much less stable than steroidal saponins, so it is of certain significance to study the biological activities of steroidal saponins of Allium. Modern pharmacological studies have demonstrated the hypoglycemic, antiplatelet aggregation, anti-inflammatory and other activities of these components through in vitro and in vivo experiments, confirming the medicinal value of Allium.

Hypoglycemic Effect
Visfatin is an insulin-mimetic adipocytokine that acts synergistically with insulin to enhance glucose uptake in vivo and in vitro and to inhibit the breakdown of liver glycogen into glucose, with insulin resistance-reducing and antidiabetic effects. Compound 133 isolated from A. macrostemon Bunge can increase the transcription of visfatin partly through the p38 MAPK pathway in differentiated 3T3-L1 adipocytes, which in turn increases the mRNA expression of visfatin and enhances the synthesis and secretion of the visfatin protein, thus having a hypoglycemic effect [47].

Antiplatelet Aggregation Effect
Adenosine diphosphate (ADP) can cause platelet aggregation through ADP receptors on platelet membranes, leading to thrombosis. Thrombus often causes angina pectoris, myocardial infarction, cerebral infarction, pulmonary embolism, and other acute diseases. A. macrostemon Bunge is a common traditional Chinese medicine used to treat cardiovascular diseases in China, as well as a common edible vegetable, and experiments have shown that the compound 88 isolated from this plant can inhibit platelet aggregation induced by ADP in vitro with an IC 50 of 0.871 mM, confirming the plausibility of the pharmacological effect of this plant [30].

Gastroprotective Effect
Compounds 177 and 181 from the bulbs of A. ampeloprasum var. porrum, a Brazilian vegetable, have been proven to have anti-gastric ulcerative effects [57,59]. They may show protective properties on gastric cells by interfering with the ulcerogenesis mechanism, protecting the gastric mucosa, and reducing gastric congestion caused by acidified ethanolinduced acute gastric injury.

Immune Adjuvant Effect
Immune adjuvants are non-specific immune-enhancing substances that are injected into the body in advance or simultaneously with antigens and enhance the response of the body to the antigen or change the type of response. The immune adjuvant effect of compound 220, which was isolated from A. ampeloprasum L. var. porrum, was higher than that of commercial adjuvants Freund's Complete Adjuvant (FCA) and Freund's Incomplete Adjuvant (FIA) in mice with ovalbumin (OVA) as an antigen by the delayedtype hypersensitivity (DTH) method [64]. The immune adjuvant effect of this compound may be due to its amphiphilic structure with hydrophilic sugar chains and lipophilic glycosides, which form adjuvant-antigen complexes and enhance antigen delivery to antigen-presenting cells for processing.

Anti-Inflammatory Effect
Ten compounds from the bulbs of A. chinense were evaluated for their anti-inflammatory effects by inhibiting NO production induced by lipopolysaccharide (LPS) in RAW 264.7 cells, and it was found that the NO inhibitory activity of steroidal saponins was related to both aglycones and sugar chains [16]. Compounds 34 and 54 showed significant anti-inflammatory effects with IC 50 values of 2.01 ± 1.40 µM and 2.49 ± 1.54 µM, respectively. The compounds 225 and 230, also from the bulbs of A. chinense showed anti-inflammatory activity with IC 50 values of 32.20 ± 0.65 µM and 34.33 ± 5.04 µM [61]. Comparison with other compounds isolated in the same period showed that the A/B ring was active in trans and inactive in cis. The carbonyl group at C-6 increased the activity, while the hydroxylation at C-24 made the activity disappear, and the sugar chain at C-3 also affected the anti-inflammatory activity.
The anti-inflammatory effects of compounds 177 and 181 from the bulbs of A. ampeloprasum var. porrum were evaluated using an in vivo model of acute inflammation formed by foot swelling caused by carrageenan gum, and both compounds showed significant anti-inflammatory effects by rapidly controlling both stages of inflammation [57,59]. PCR was used to detect the inhibitory effects of compounds 37, 38, and 39 from the bulbs of A. macrostemon Bunge on the expression of CD40 ligand (CD40L) on the surface of platelet membranes activated by ADP stimulation [18]. Compounds 37 and 38 were able to significantly inhibit CD40L expression in a dose-dependent manner, indicating that they could be used as CD40L inhibitors for the treatment of platelet inflammation. Figure 5 presents the effect of the structure-activity relationship of steroidal saponins on the anti-inflammatory effect.

Cytotoxicity and Antitumor Effects
Studies have shown that some steroidal saponins from Allium are cytotoxic to tumor cells and may serve as drug candidates to treat cancer. Currently, it has been reported that steroidal saponins have significant antitumor activity on more than twenty types of tumor cells, such as A549 (human lung cancer cells), AGS (human gastric gland cancer cells), CNE-1 (human nasopharyngeal cancer cells), DLD-1 (human colorectal cancer cells), HeLa (human cervical cancer cells), HepG2 (human liver cancer cells), HT-29 (human colon cancer cells), MCF-7 (human breast cancer cells), etc.
The cytotoxicity of steroidal saponins is influenced by both aglycone and sugar chains. Compounds 111, 155, and 247 of spirostane saponins and compounds 259 and 260 of cholestane saponins from the bulbs of A. porrum L. exhibited strong cytotoxicity against both J-774 (IC50 3.7, 2.1, 5.8, 4.6, and 4.0 μg/mL, respectively) and WEHI-164 cells (IC50 4.8, 1.9, 4.3, 5.8, and 5.4 μg/mL, respectively), although a lack of activity of cholestane saponin was previously reported in the literature [36]. Compounds 111, 114, and 201 from the flowers of A. porrum L. were cytotoxic to mouse peritoneal cells, with an IC50 ranging from 5.70 to 11.13 μM [37]. Compounds 133,134,137,138,198,199, and 216 from the bulbs of A. chinense exhibited significant cytotoxicity against HepG2, A549, SPC-A-1 (human lung adenocarcinoma cells), CNE-1, and MGC80-3 (human gastric adenocarcinoma cells), with an IC50 ranging from 1.43 to 22.32 μM [61]. Compound 199 was not cytotoxic to MRC-5 (human embryonic lung fibroblasts), and its antitumor effects were selective. Comparison with other compounds isolated at the same time reveals that the configuration of H at C-5 and the selective acetylation of the hydroxyl group of sugar significantly affected the cytotoxicity. Beyond that, the polar oxygen-containing group (C=O) at C-6 of the B ring and the hydroxyl group at C-24 of the F ring led to a decrease in antitumor activity. Compounds 34, 54, 55, and 56, also from the bulbs of A. chinense, showed significant inhibitory effects on HepG2,2 A549, SPC-A-1, MGC80-3, MDA-MB-231(human breast cancer cells), SW620 (human colon cancer cell), and CNE-1, with IC50 values ranging from 1.76 to 22.83 μM [16]. Compound 54 also exhibited selective inhibitory effects and showed no cytotoxicity to MRC-5, and it could exert antitumor effects by inducing HepG2 cell cycle arrest and apoptosis. The relationship between steroidal saponins' cytotoxicity on normal cells and the pharmacological activities, such as anti-inflammation and immune enhancement, deserves more in-depth study. Figure 6 presents the effect of the structure-activity relationship of steroidal saponins on cytotoxicity.

Cytotoxicity and Antitumor Effects
Studies have shown that some steroidal saponins from Allium are cytotoxic to tumor cells and may serve as drug candidates to treat cancer. Currently, it has been reported that steroidal saponins have significant antitumor activity on more than twenty types of tumor cells, such as A549 (human lung cancer cells), AGS (human gastric gland cancer cells), CNE-1 (human nasopharyngeal cancer cells), DLD-1 (human colorectal cancer cells), HeLa (human cervical cancer cells), HepG2 (human liver cancer cells), HT-29 (human colon cancer cells), MCF-7 (human breast cancer cells), etc.
The cytotoxicity of steroidal saponins is influenced by both aglycone and sugar chains.  [36]. Compounds 111, 114, and 201 from the flowers of A. porrum L. were cytotoxic to mouse peritoneal cells, with an IC 50 ranging from 5.70 to 11.13 µM [37]. Compounds 133,134,137,138,198,199, and 216 from the bulbs of A. chinense exhibited significant cytotoxicity against HepG2, A549, SPC-A-1 (human lung adenocarcinoma cells), CNE-1, and MGC80-3 (human gastric adenocarcinoma cells), with an IC 50 ranging from 1.43 to 22.32 µM [61]. Compound 199 was not cytotoxic to MRC-5 (human embryonic lung fibroblasts), and its antitumor effects were selective. Comparison with other compounds isolated at the same time reveals that the configuration of H at C-5 and the selective acetylation of the hydroxyl group of sugar significantly affected the cytotoxicity. Beyond that, the polar oxygen-containing group (C=O) at C-6 of the B ring and the hydroxyl group at C-24 of the F ring led to a decrease in antitumor activity. Compounds 34, 54, 55, and 56, also from the bulbs of A. chinense, showed significant inhibitory effects on HepG2,2 A549, SPC-A-1, MGC80-3, MDA-MB-231(human breast cancer cells), SW620 (human colon cancer cell), and CNE-1, with IC 50 values ranging from 1.76 to 22.83 µM [16]. Compound 54 also exhibited selective inhibitory effects and showed no cytotoxicity to MRC-5, and it could exert antitumor effects by inducing HepG2 cell cycle arrest and apoptosis. The relationship between steroidal saponins' cytotoxicity on normal cells and the pharmacological activities, such as anti-inflammation and immune enhancement, deserves more in-depth study. Figure 6 presents the effect of the structure-activity relationship of steroidal saponins on cytotoxicity.

Antimicrobial Effect
Steroidal saponins usually have the effect of inhibiting fungi and bacteria. Several studies have investigated the antifungal effect of steroidal saponins isolated from Allium. The reported fungal species include Aspergillus niger, Alternaria alternata, Alternaria porri, Botrytis cinerea, Candida albicans, Fusarium solani, Fusarium oxysporum, Fusarium oxysporum f. sp. lycopersici, Mortierella ramanniana, Penicillium italicum, Pythium ultimum, Rhizoctonia solani, Trichoderma harzianum, Trichoderma harzianum (strains P1), and Trichoderma harzianum (strain T39). Compared to fungi, bacteria are usually less sensitive to saponin components, and fewer species of bacteria have been reported to be inhibited by steroidal saponins from Allium, including Bacillus subtilis, Escherichia coli, etc.
Compounds 29, 30, 116, 140, and 175 from the bulbs of Allium minutiflorum Regel inhibited ten soil-borne fungal pathogens, including Fusarium oxysporum, Fusarium oxysporum f. sp. lycopersici, Fusarium solani, and biocontrol fungi, but not against bacteria such as Xanthomonas campestris pv. Campestris, Agrobacterium tumefaciens, and Streptomyces turgidiscabies [14]. Compound 175, with a high content in bulbs (83.5 mg/kg), showed the strongest antifungal activity. A structure-activity relationship analysis revealed that the spirostane skeleton was more active than the furostane skeleton, and the hydroxyl group at C-5 of the furostane saponins promoted antifungal activity. Compounds 187 and 188 from the seeds of Allium ampeloprasum subsp. Persicum showed antifungal activity against Penicillium italicum, Aspergillus niger, and Trichoderma harzianum, while other steroidal saponins isolated at the same time showed no antifungal activity, indicating that the spirostane skeleton was more active than the furostane and cholestane skeletons [21]. This also confirms that the hydroxyl group at C-6 could enhance the antifungal activity.
Six spirostane saponins from the roots of Allium tuberosum were evaluated for their antibacterial effects against Bacillus subtilis and Escherichia coli, and the results showed that compound 192 exhibited good antibacterial activity (MIC 64 μg/mL, both), compounds 194 (MIC 16 and 32 μg/mL, respectively) and 195 (MIC 16 μg/mL, both) showed potent antibacterial activity, while compounds 167, 191, and 193 showed almost no bacterial inhibitory effect (MIC > 128 μg/mL) [55]. A structure-activity relationship analysis revealed that the sugar at C-3 and the hydroxyl group at C-19 enhanced the antibacterial activity, while the hydroxyl groups at C-2 and C-5 attenuated the antibacterial activity. Figure 7 presents the effects of steroidal saponins on antifungal and antibacterial activities, respectively.

Antimicrobial Effect
Steroidal saponins usually have the effect of inhibiting fungi and bacteria. Several studies have investigated the antifungal effect of steroidal saponins isolated from Allium. The reported fungal species include Aspergillus niger, Alternaria alternata, Alternaria porri, Botrytis cinerea, Candida albicans, Fusarium solani, Fusarium oxysporum, Fusarium oxysporum f. sp. lycopersici, Mortierella ramanniana, Penicillium italicum, Pythium ultimum, Rhizoctonia solani, Trichoderma harzianum, Trichoderma harzianum (strains P1), and Trichoderma harzianum (strain T39). Compared to fungi, bacteria are usually less sensitive to saponin components, and fewer species of bacteria have been reported to be inhibited by steroidal saponins from Allium, including Bacillus subtilis, Escherichia coli, etc.
Compounds 29, 30, 116, 140, and 175 from the bulbs of Allium minutiflorum Regel inhibited ten soil-borne fungal pathogens, including Fusarium oxysporum, Fusarium oxysporum f. sp. lycopersici, Fusarium solani, and biocontrol fungi, but not against bacteria such as Xanthomonas campestris pv. Campestris, Agrobacterium tumefaciens, and Streptomyces turgidiscabies [14]. Compound 175, with a high content in bulbs (83.5 mg/kg), showed the strongest antifungal activity. A structure-activity relationship analysis revealed that the spirostane skeleton was more active than the furostane skeleton, and the hydroxyl group at C-5 of the furostane saponins promoted antifungal activity. Compounds 187 and 188 from the seeds of Allium ampeloprasum subsp. Persicum showed antifungal activity against Penicillium italicum, Aspergillus niger, and Trichoderma harzianum, while other steroidal saponins isolated at the same time showed no antifungal activity, indicating that the spirostane skeleton was more active than the furostane and cholestane skeletons [21]. This also confirms that the hydroxyl group at C-6 could enhance the antifungal activity.
Six spirostane saponins from the roots of Allium tuberosum were evaluated for their antibacterial effects against Bacillus subtilis and Escherichia coli, and the results showed that compound 192 exhibited good antibacterial activity (MIC 64 µg/mL, both), compounds 194 (MIC 16 and 32 µg/mL, respectively) and 195 (MIC 16 µg/mL, both) showed potent antibacterial activity, while compounds 167, 191, and 193 showed almost no bacterial inhibitory effect (MIC > 128 µg/mL) [55]. A structure-activity relationship analysis revealed that the sugar at C-3 and the hydroxyl group at C-19 enhanced the antibacterial activity, while the hydroxyl groups at C-2 and C-5 attenuated the antibacterial activity. Figure 7 presents the effects of steroidal saponins on antifungal and antibacterial activities, respectively.

Enzyme Activity Inhibition Effect
Na,K-ATPase is responsible for the active transport of Na + and K + in cells, and drugs that inhibit Na,K-ATPase activity can be used to treat diseases associated with ion active transport disorders for which the transporter enzyme is responsible. Fourteen compounds from the collective fruit of A. karataviense Rgl and A. cepa L were tested for their inhibition of Na,K-ATPase activity [62]. Compounds 202, 203, and 204 had the strongest enzyme inhibitory activity, with IC50 values of 1.0 × 10 −5 M, 3.4 × 10 −5 M, and 8.8 × 10 −5 M. Furthermore, compounds 202 and 203 had non-competitive inhibition, and compound 204 had competitive inhibition. The results of the structure-activity analysis show that the hydroxyl group at C-24 of the F ring decreased the inhibitory activity, the carbonyl group at C-6 of the steroid skeleton slightly enhanced the inhibitory activity, and when the F ring was opened and the sugar was connected to the hydroxyl group at C-25, the inhibitory activity was weakened, and even enzyme activity was promoted. In addition, compounds 133 and 134 from the bulbs of A. chinense also inhibited Na, K-ATPase, with an IC50 of 4 × 10 −5 M [46]. cAMP phosphodiesterases are enzymes that catabolize cyclic adenosine acids under the activation of calmodulin bound to Ca 2+ . The inhibition of cAMP phosphodiesterase increases intracellular cAMP content, which enhances myocardial contraction, dilates peripheral blood vessels, and improves heart failure. Compounds 133, 134, 226, 227, and 228 from the bulbs of A. chinense significantly reduced the activity of cAMP phosphodiesterase, especially compound 227, with an IC50 of 3.3 × 10 −5 M, which is comparable to the positive drug papaverine (IC50 3.0 × 10 −5 M) [46]. The compounds 7, 12, 13, 114, 117, 128, and 130 from the bulbs of A. giganteum can significantly inhibit the activity of cAMP phosphodiesterase [6]. A structure-activity relationship analysis revealed that the inhibition activity was enhanced when the (S)-3-hydroxy-3-methylglutaroyl (HMG group) was attached to the hydroxyl group at C-4 of xylose. Furostane steroidal saponins 7, 12, and 13 exhibited stronger activity than the corresponding spirostane steroidal saponins 114 and 117, which may be related to the multiple hydroxyl groups in their A and B rings.

Antispasmodic Effect
Compounds 27, 28, and 172 isolated from the flowers of A. hirtifolium Boiss and compounds 15, 16, 19, 20, and 170 isolated from the bulbs of A. elburzense were tested for their antispasmodic activity by using the histamine-induced contractions of isolated ileum of guinea-pig as a model [12]. Compounds 19, 20, and 170 exhibited the strongest inhibitory contractile activity. A structure-activity relationship analysis showed that the antispasmodic activity can be enhanced by the hydroxyl group at C-5 and glucose at C-26, while it can be attenuated by the hydroxyl group at C-6 and glucose at C-3.

Enzyme Activity Inhibition Effect
Na,K-ATPase is responsible for the active transport of Na + and K + in cells, and drugs that inhibit Na,K-ATPase activity can be used to treat diseases associated with ion active transport disorders for which the transporter enzyme is responsible. Fourteen compounds from the collective fruit of A. karataviense Rgl and A. cepa L were tested for their inhibition of Na,K-ATPase activity [62]. Compounds 202, 203, and 204 had the strongest enzyme inhibitory activity, with IC 50 values of 1.0 × 10 −5 M, 3.4 × 10 −5 M, and 8.8 × 10 −5 M. Furthermore, compounds 202 and 203 had non-competitive inhibition, and compound 204 had competitive inhibition. The results of the structure-activity analysis show that the hydroxyl group at C-24 of the F ring decreased the inhibitory activity, the carbonyl group at C-6 of the steroid skeleton slightly enhanced the inhibitory activity, and when the F ring was opened and the sugar was connected to the hydroxyl group at C-25, the inhibitory activity was weakened, and even enzyme activity was promoted. In addition, compounds 133 and 134 from the bulbs of A. chinense also inhibited Na, K-ATPase, with an IC 50 of 4 × 10 −5 M [46]. cAMP phosphodiesterases are enzymes that catabolize cyclic adenosine acids under the activation of calmodulin bound to Ca 2+ . The inhibition of cAMP phosphodiesterase increases intracellular cAMP content, which enhances myocardial contraction, dilates peripheral blood vessels, and improves heart failure. Compounds 133, 134, 226, 227, and 228 from the bulbs of A. chinense significantly reduced the activity of cAMP phosphodiesterase, especially compound 227, with an IC 50 of 3.3 × 10 −5 M, which is comparable to the positive drug papaverine (IC 50 3.0 × 10 −5 M) [46]. The compounds 7, 12, 13, 114, 117, 128, and 130 from the bulbs of A. giganteum can significantly inhibit the activity of cAMP phosphodiesterase [6]. A structure-activity relationship analysis revealed that the inhibition activity was enhanced when the (S)-3hydroxy-3-methylglutaroyl (HMG group) was attached to the hydroxyl group at C-4 of xylose. Furostane steroidal saponins 7, 12, and 13 exhibited stronger activity than the corresponding spirostane steroidal saponins 114 and 117, which may be related to the multiple hydroxyl groups in their A and B rings.

Antispasmodic Effect
Compounds 27, 28, and 172 isolated from the flowers of A. hirtifolium Boiss and compounds 15, 16, 19, 20, and 170 isolated from the bulbs of A. elburzense were tested for their antispasmodic activity by using the histamine-induced contractions of isolated ileum of guinea-pig as a model [12]. Compounds 19, 20, and 170 exhibited the strongest inhibitory contractile activity. A structure-activity relationship analysis showed that the antispasmodic activity can be enhanced by the hydroxyl group at C-5 and glucose at C-26, while it can be attenuated by the hydroxyl group at C-6 and glucose at C-3.

Cardiomyocyte Regulation Effect
Calcium ions play an important role in the regulation of cell physiological function. Under normal physiological conditions, the intracellular calcium levels are relatively stable and in dynamic regulation; otherwise, it may lead to cell damage or apoptosis. Compounds 33, 35, 36, and 90 isolated from A. macrostemon further increased KCl-induced [Ca 2+ ] i increase in guinea pig cardiomyocytes, suggesting that it may enhance myocardial function [17]. In contrast, compound 87 inhibited the KCl-induced [Ca 2+ ] i increase, suggesting its potential for the treatment of heart failure.

Nerve Cell Protection Effect
The antioxidant activity was investigated using the CCK-8 method for six compounds isolated from A. chinense G. Don, and hydrogen peroxide was selected to induce a neuronal oxidative damage model in PC12 cells [13]. Compared to five other compounds isolated simultaneously, compound 57 had a significant protective effect on neuronal cell injury induced by hydrogen peroxide in a dose-dependent manner, which may be related to its H at C-22 and the higher amounts of glucose and galactose in the sugar chain.

Hemolysis Effect
Steroidal saponins can bind to cholesterol on the erythrocyte membrane to form insoluble complexes, disrupting the osmotic pressure of the cell membrane and inducing hemolysis. Usually, spirostane saponins have stronger hemolytic activity due to a higher affinity with cholesterol. Compounds 177, 181, and 220 isolated from the bulbs of A. ampeloprasum var. porrum, have all shown hemolytic ability, with HD 50 values of 20 µg/mL, 6.5 µg/mL, and 13 µg/mL, respectively [57,59,64]. Their hemolysis is on the same order of magnitude as that of the reference commercial saponin QS-21 isolated from Quillaja saponaria (HD 50 5 µg/mL).

Proposed Biosynthetic Pathways of Allium Steroidal Saponins
The biosynthetic pathway of steroid saponins is acetyl coenzyme A through the mevalonate (MVA) pathway or pyruvate through the methylerythritol 4-phosphate (MEP) pathway to produce farnesyl pyrophosphate (FPP), which is then catalyzed by squalene synthase (SQS) to produce squalene, and by squalene epoxidase (SQE) to produce 2, 3-oxidized squalene, and then catalyzed by cycloatenol synthase (CAS) to produce cycloatenol, which is the precursor of steroid compounds. The cycloatenol further synthesizes cholestanol, which is then hydroxylated at C-22, C-26, and C-16 to form semi-ketal structure compounds, which in turn synthesizes furostane saponin. The glycosidic bond at C-26 of furostane saponin is easily hydrolyzed, and after hydrolysis, it cyclizes into a spiral ketal structure to synthesize spirostane saponin.
Cholestane compounds 259 and 260 can be produced by the glycosylation of 1,16,22trihydroxycholestrol. Pregnane compound 274 can be synthesized from cholesterol by multi-step oxidative breakage to produce pregnenolone, which in turn synthesizes compound 274 by glycosylation. Compound 271 can be synthesized from furostane compound 24 by oxidative breakage at C-22, and compound 272 can be synthesized from furostane compound 35 by a similar mechanism. The oxygen bridge of the A-ring of spirostane compound 253 can be synthesized by the dehydration cyclization of compound 116. Compound 269 can be synthesized from compound 141 by oxidation at C-2 and C-3, and then C-3 is dehydrated with C-6 to form a lactone ring. The proposed biosynthetic pathways of Allium steroidal saponin are shown in Figure 8.

Conclusions
In recent decades, as common functional vegetables, a wide variety of Allium plants have been extensively studied for their important edible and medicinal values. Undoubtedly, their secondary metabolite, a steroidal saponin, is one of the most important chemical bases for the healthcare functions of Allium. In this paper, we have summarized the chemical constituents, biological activities, and structure-activity relationships of steroidal saponins isolated from Allium and have proposed the biosynthetic pathways of some key compounds to clarify the molecular basis of the rich health function of Allium as a vegetable and condiment from the perspective of secondary metabolites. In addition, we have explored the positive role of Allium in the prevention and treatment of diseases more comprehensively.